Measuring Team Hierarchy During High-Stakes Clinical Decision Making: Development and Validation of a New Behavioral Observation Method.

PURPOSE: Hierarchy is often cited as a cause of health care team failure; however, there are no validated measures of team hierarchy. Research on group processes in sociology provides a theoretical framework-status characteristics and expectation states (SCES)-that explains the mechanisms that produce the observable power and prestige order (status hierarchy) of the team. The authors use this formal theoretical framework to gather evidence of validity by adapting the method to measure the status hierarchy of medical teams. METHOD: In this retrospective, secondary analysis, the authors analyzed archived videorecorded training exercises conducted between 2007 and 2010 of mixed-gender health care teams of first-year residents and nurses engaged in simulated, complex decision-making scenarios. Analyses were conducted in 2013 with data reanalyzed in July 2022. By adapting the SCES framework for the unique features of academic health care, they developed and refined a coding method from videos and transcripts. To examine validity, they consider the content, response process, internal structure, relation to other variables, and consequences of the framework. RESULTS: Having established an acceptable level of coding reliability for key variables for videos and transcripts, the authors demonstrate relation to other variables, specifically detailing how the coding scheme delineates 2 status characteristics-occupation and gender. The mean numbers of statement types by gender and occupation were largely as predicted. Directives, question directives, patient work, and knowledge claims were more likely to be coded during video than transcript coding, whereas questions, statements of fact, and compliance were more likely to be coded during transcript than video coding. However, the relative rates of each statement type by status remained largely consistent among the coding methods. CONCLUSIONS: This study provides important insight into the mechanisms by which hierarchy impacts team decision making and develops the necessary framework and measurement tool to perform larger studies.

Patient and Clinician Perceptions of Factors Relevant to Ideal Specialty Consultations.

Importance: Inpatient subspecialty consultations, a common and expensive practice within inpatient medicine, do not always go well; however, little is known about the failure modes of consultation, thus making it difficult to identify interventions to improve consultation quality. Objective: To understand how stakeholders envision the ideal inpatient consultation and identify how and why consultations commonly fall short of this ideal. Design, Setting, and Participants: This qualitative study used in-depth, semistructured interviews collected from April to October 2017 and analyzed from January 2018 to February 2020 using conventional content analysis. The setting was a single academic medical center in Boston, Massachusetts. Participants were hospitalists and specialists who had requested or performed a consultation for a non-intensive care unit patient in the previous 4 months, patients who had received a consultation while hospitalized at the medical center in the previous 15 months, and family members of such patients. Main Outcomes and Measures: Consultation experiences reported by participants. Clinicians were asked about characteristics of the ideal consultation, positive and negative consultation experiences, costs and benefits, and suggested improvements. Patients and family members were asked about their consultation experience, changes in care, communication preferences, and suggested improvements. Results: The study included 38 participants: 17 specialists, 13 hospitalists, 4 patients, and 4 family members. More than half (21 of 38) of the participants were female. There were 11 key information exchanges identified that occur among the specialist team, primary team, and patient/family during an ideal consultation. These exchanges are time sensitive and primarily carried out through unwritten protocols. We also identified 6 defects (process failures) that commonly derail information exchanges (complete omission, exclusion of a key stakeholder, poor timing, incomplete or inaccurate information, and misinterpretation) and 5 contextual factors (roles and boundaries, professionalism, team hierarchy, availability, and operational know-how) that influence how information exchange unfolds, making some consultations more prone to defects. Conclusions and Relevance: Successful inpatient consultation requires a complicated, sequenced series of time-sensitive information exchanges that are highly vulnerable to failure. Maximizing the benefit of consultations will likely entail not only minimizing low-value consultations but also actively preventing defects, such as information inaccuracies and misinterpretation, that commonly derail the consultation process.

Comparison of sequencing data processing pipelines and application to underrepresented African human populations.

BACKGROUND: Population genetic studies of humans make increasing use of high-throughput sequencing in order to capture diversity in an unbiased way. There is an abundance of sequencing technologies, bioinformatic tools and the available genomes are increasing in number. Studies have evaluated and compared some of these technologies and tools, such as the Genome Analysis Toolkit (GATK) and its "Best Practices" bioinformatic pipelines. However, studies often focus on a few genomes of Eurasian origin in order to detect technical issues. We instead surveyed the use of the GATK tools and established a pipeline for processing high coverage full genomes from a diverse set of populations, including Sub-Saharan African groups, in order to reveal challenges from human diversity and stratification. RESULTS: We surveyed 29 studies using high-throughput sequencing data, and compared their strategies for data pre-processing and variant calling. We found that processing of data is very variable across studies and that the GATK "Best Practices" are seldom followed strictly. We then compared three versions of a GATK pipeline, differing in the inclusion of an indel realignment step and with a modification of the base quality score recalibration step. We applied the pipelines on a diverse set of 28 individuals. We compared the pipelines in terms of count of called variants and overlap of the callsets. We found that the pipelines resulted in similar callsets, in particular after callset filtering. We also ran one of the pipelines on a larger dataset of 179 individuals. We noted that including more individuals at the joint genotyping step resulted in different counts of variants. At the individual level, we observed that the average genome coverage was correlated to the number of variants called. CONCLUSIONS: We conclude that applying the GATK "Best Practices" pipeline, including their recommended reference datasets, to underrepresented populations does not lead to a decrease in the number of called variants compared to alternative pipelines. We recommend to aim for coverage of > 30X if identifying most variants is important, and to work with large sample sizes at the variant calling stage, also for underrepresented individuals and populations.

Copy number determination of the gene for the human pancreatic polypeptide receptor NPY4R using read depth analysis and droplet digital PCR.

BACKGROUND: Copy number variation (CNV) plays an important role in human genetic diversity and has been associated with multiple complex disorders. Here we investigate a CNV on chromosome 10q11.22 that spans NPY4R, the gene for the appetite-regulating pancreatic polypeptide receptor Y4. This genomic region has been challenging to map due to multiple repeated elements and its precise organization has not yet been resolved. Previous studies using microarrays were interpreted to show that the most common copy number was 2 per genome. RESULTS: We have investigated 18 individuals from the 1000 Genomes project using the well-established method of read depth analysis and the new droplet digital PCR (ddPCR) method. We find that the most common copy number for NPY4R is 4. The estimated number of copies ranged from three to seven based on read depth analyses with Control-FREEC and CNVnator, and from four to seven based on ddPCR. We suggest that the difference between our results and those published previously can be explained by methodological differences such as reference gene choice, data normalization and method reliability. Three high-quality archaic human genomes (two Neanderthal and one Denisova) display four copies of the NPY4R gene indicating that a duplication occurred prior to the human-Neanderthal/Denisova split. CONCLUSIONS: We conclude that ddPCR is a sensitive and reliable method for CNV determination, that it can be used for read depth calibration in CNV studies based on already available whole-genome sequencing data, and that further investigation of NPY4R copy number variation and its consequences are necessary due to the role of Y4 receptor in food intake regulation.

Measuring the quality of inpatient specialist consultation in the intensive care unit: Nursing and family experiences of communication.

RATIONALE: Critically ill patients in the intensive care unit (ICU) often require the care of specialist physicians for clinical or procedural expertise. The current state of communication between specialist physicians and families and nurses has not been explored. OBJECTIVES: To document the receipt of communication by nurses and family members regarding consultations performed on their patient or loved one, and to quantify how this impacts their overall perceptions of the quality of specialty care. METHODS: Prospective survey of 60 adult family members and 90 nurses of 189 ICU patients who received a specialist consultation between March and October of 2015 in a single academic medical center in the United States. Surveys measured the prevalence of direct communication-defined as communication conducted in person, via telephone, or via text-page in which the specialist team gathered information about the patient from the nurse/family member and/or shared recommendations for care-and perceived quality of care. RESULTS: In about two-thirds of family surveys (40/60) and one-half of nurse surveys (75/160), respondents had no direct communication with the specialist team that performed the consultation. Compared to nurses who had no direct communication with the specialists, those who did were 1.5 times more likely to rate the consultation as "excellent" (RR 1.48, 95% CI 1.2-1.8, p<0.001). Nearly 40% (22/60) of families knew so little about the consultation that they felt incapable of evaluating it. CONCLUSIONS: Most ICU families and nurses have no interaction with specialist providers. Nurses' frequent exclusion from conversations about specialty care may pose safety risks and increase the likelihood of mixed messages for patients and families, most of whom desire some interaction with specialists. Future research is needed to identify effective mechanisms for information sharing that keep nurses and families aware of consultation requests, delivery, and outcomes without increasing the risk of mixed messages.

Eliciting stakeholder preferences for patient-centered research.

BACKGROUND: Rather than identifying exposures and outcomes for research solely based on interests of medical professionals, there is a need for research that answers questions that are important to patients, so that they may make treatment decisions based on evidence that reflect their individual preferences. OBJECTIVE: To identify exposures and outcomes of interest that could be studied with electronic health record data from inpatient care. DESIGN SETTING PARTICIPANTS: Mixed-methods analysis of semi-structured interviews administered in 2017 to 76 patients and 26 physicians who receive or provide care at Beth Israel Deaconess Medical Center in Boston, MA. MEASUREMENTS: After conducting detailed semi-structured interviews about topics of interest that can be studied using electronic health records of inpatient care, we used an inductive approach to identify themes about the health care experience. RESULTS: Participants reported concerns about adverse effects of medication changes, drug interactions, and surgery and other invasive procedures. The outcomes of greatest concern to them were in-hospital deaths and hospital-acquired infections. Participants commented on the importance of clear communication and information transfers, the hospital environment, accurate skills and knowledge, and upholding patient dignity and respect. CONCLUSION: Engaging patients and physicians in the research development process provided insight to the exposures and outcomes they consider important. Our questions about exposures and outcomes of interest were restricted to topics that could be studied with electronic health record data from inpatient care, but using a similar approach to elicit feedback about the health care experience could be used to glean insight for other areas of future research.

Clinician Perspectives on an Electronic Portal to Improve Communication with Patients and Families in the Intensive Care Unit.

RATIONALE: Communication in the intensive care unit (ICU) often falls short of patient and family needs, putting them at risk for significant physical and emotional harm. As electronic patient portals rapidly evolve, one designed specifically for the ICU might potentially enhance communication among patients, family members, and clinicians; however, the views of frontline ICU staff on such technology are unknown. OBJECTIVES: To identify clinician perspectives on the current state of communication among patients, families, and clinicians in the ICU, and assess their views on whether and how an electronic portal may address existing communication deficits and improve care. METHODS: Three focus groups comprised altogether of 26 clinicians from 6 ICUs, representing several disciplines in an academic medical center in Boston, Massachusetts. Transcripts were analyzed inductively for major themes using grounded theory. MEASUREMENTS AND MAIN RESULTS: We identified seven themes reflecting clinician perspectives on communication challenges and desired portal functionality: (1) comprehension and literacy; (2) results and updates; (3) patient and family preferences; (4) interclinician communication; (5) family informational needs; (6) the ICU as an unfamiliar environment; and (7) enhancing humanism through technology. Each theme included current gaps in practice, potential benefits and concerns related to an ICU communication portal, and participant recommendations. Benefits included enhanced education, patient/family engagement, and clinician workflow. Challenges included the stress and uncertainty of ICU care, fear of technology replacing human connection, existing interclinician communication failures, and the tension between informing families without overwhelming them. CONCLUSIONS: Overall, clinicians were cautiously supportive of an electronic portal to enhance communication in the ICU and made several specific recommendations for design and implementation. As new technologies expand opportunities for greater transparency and participation in care, clinician buy-in and positive impact will depend, in large part, on the extent to which the concerns of stakeholders are addressed. At the same time, clinicians anticipate several potential benefits that could help support provider workflow and engage patients and families through enhanced communication and humanism.

Abolished InsP3R2 function inhibits sweat secretion in both humans and mice.

There are 3 major sweat-producing glands present in skin; eccrine, apocrine, and apoeccrine glands. Due to the high rate of secretion, eccrine sweating is a vital regulator of body temperature in response to thermal stress in humans; therefore, an inability to sweat (anhidrosis) results in heat intolerance that may cause impaired consciousness and death. Here, we have reported 5 members of a consanguineous family with generalized, isolated anhidrosis, but morphologically normal eccrine sweat glands. Whole-genome analysis identified the presence of a homozygous missense mutation in ITPR2, which encodes the type 2 inositol 1,4,5-trisphosphate receptor (InsP3R2), that was present in all affected family members. We determined that the mutation is localized within the pore forming region of InsP3R2 and abrogates Ca2+ release from the endoplasmic reticulum, which suggests that intracellular Ca2+ release by InsP3R2 in clear cells of the sweat glands is important for eccrine sweat production. Itpr2-/- mice exhibited a marked reduction in sweat secretion, and evaluation of sweat glands from Itpr2-/- animals revealed a decrease in Ca2+ response compared with controls. Together, our data indicate that loss of InsP3R2-mediated Ca2+ release causes isolated anhidrosis in humans and suggest that specific InsP3R inhibitors have the potential to reduce sweat production in hyperhidrosis.

Elements of a high-quality inpatient consultation in the intensive care unit. A qualitative study.

RATIONALE: Inpatient consultation by specialists is one of the most common medical interventions in the modern intensive care unit (ICU), but few data exist on components of high-quality consultation. OBJECTIVES: Our objective was to use qualitative methods to develop a conceptual framework of consultative quality in critically ill patients. METHODS: We conducted a qualitative study of medical ICU physicians at a single institution using a novel, semistructured interview guide. We elicited physicians' attitudes toward processes of obtaining specialty consultation, identified perceived elements of high-quality consults, and identified barriers to obtaining high-quality consults. We used grounded theory to identify themes. MEASUREMENTS AND MAIN RESULTS: ICU physicians described four common reasons for involving a consulting physician: the need for clinical or procedural expertise, an explicit or implicit protocol of the institution mandating the consult, an opportunity to provide education to the primary or consulting team, and/or at the family's request. Participants identified seven components of a high-quality consult, including the consulting teams' (1) decisiveness, (2) thoroughness, (3) level of interest, (4) professionalism, (5) expertise, (6) timeliness, and (7) involvement with the family of the patient. The intensive care team, the consult team, the health system, and the temporal context in which the consultation takes place may influence the quality of the consultation. CONCLUSIONS: Several key factors are necessary for a consult to be judged high quality. An opportunity exists to develop an instrument to assess and to improve specialty consultations in the ICU based on these findings.

Welander distal myopathy caused by an ancient founder mutation in TIA1 associated with perturbed splicing.

Welander distal myopathy (WDM) is an adult onset autosomal dominant disorder characterized by distal limb weakness, which progresses slowly from the fifth decade. All WDM patients are of Swedish or Finnish descent and share a rare chromosome 2p13 haplotype. We restricted the WDM-associated haplotype followed by whole exome sequencing. Within the conserved haplotype, we identified a single heterozygous mutation c.1150G>A (p.E384K) in T-cell intracellular antigen-1 (TIA1) in all WDM patients investigated (n = 43). The TIA1 protein regulates splicing, and translation through direct interaction with mRNA and the p.E384K mutation is located in the C-terminal Q-rich domain that interacts with the U1-C splicing factor. TIA1 has been shown to prevent skipping of SMN2 exon 7, and we show that WDM patients have increased levels of spliced SMN2 in skeletal muscle cells when compared with controls. Immunostaining of WDM muscle biopsies showed accumulation of TIA1 and stress granulae proteins adjacent to intracellular inclusions, a typical finding in WDM. The combined findings strongly suggest that the TIA1 mutation causes perturbed RNA splicing and cellular stress resulting in WDM. The selection against the mutation is likely to be negligible and the age of the TIA1 founder mutation was calculated to approximately 1,050 years, which coincides with the epoch of early seafaring across the Baltic Sea.

Genome-wide sequencing for the identification of rearrangements associated with Tourette syndrome and obsessive-compulsive disorder.

BACKGROUND: Tourette Syndrome (TS) is a neuropsychiatric disorder in children characterized by motor and verbal tics. Although several genes have been suggested in the etiology of TS, the genetic mechanisms remain poorly understood. METHODS: Using cytogenetics and FISH analysis, we identified an apparently balanced t(6,22)(q16.2;p13) in a male patient with TS and obsessive-compulsive disorder (OCD). In order to map the breakpoints and to identify additional submicroscopic rearrangements, we performed whole genome mate-pair sequencing and CGH-array analysis on DNA from the proband. RESULTS: Sequence and CGH array analysis revealed a 400 kb deletion located 1.3 Mb telomeric of the chromosome 6q breakpoint, which has not been reported in controls. The deletion affects three genes (GPR63, NDUFA4 and KLHL32) and overlaps a region previously found deleted in a girl with autistic features and speech delay. The proband's mother, also a carrier of the translocation, was diagnosed with OCD and shares the deletion. We also describe a further potentially related rearrangement which, while unmapped in Homo sapiens, was consistent with the chimpanzee genome. CONCLUSIONS: We conclude that genome-wide sequencing at relatively low resolution can be used for the identification of submicroscopic rearrangements. We also show that large rearrangements may escape detection using standard analysis of whole genome sequencing data. Our findings further provide a candidate region for TS and OCD on chromosome 6q16.

Genetic adaptation of fatty-acid metabolism: a human-specific haplotype increasing the biosynthesis of long-chain omega-3 and omega-6 fatty acids.

Omega-3 and omega-6 long-chain polyunsaturated fatty acids (LC-PUFAs) are essential for the development and function of the human brain. They can be obtained directly from food, e.g., fish, or synthesized from precursor molecules found in vegetable oils. To determine the importance of genetic variability to fatty-acid biosynthesis, we studied FADS1 and FADS2, which encode rate-limiting enzymes for fatty-acid conversion. We performed genome-wide genotyping (n = 5,652 individuals) and targeted resequencing (n = 960 individuals) of the FADS region in five European population cohorts. We also analyzed available genomic data from human populations, archaic hominins, and more distant primates. Our results show that present-day humans have two common FADS haplotypes-defined by 28 closely linked SNPs across 38.9 kb-that differ dramatically in their ability to generate LC-PUFAs. No independent effects on FADS activity were seen for rare SNPs detected by targeted resequencing. The more efficient, evolutionarily derived haplotype appeared after the lineage split leading to modern humans and Neanderthals and shows evidence of positive selection. This human-specific haplotype increases the efficiency of synthesizing essential long-chain fatty acids from precursors and thereby might have provided an advantage in environments with limited access to dietary LC-PUFAs. In the modern world, this haplotype has been associated with lifestyle-related diseases, such as coronary artery disease.

Characterizing and interpreting genetic variation from personal genome sequencing.

Since the completion of the human genome project, there has been enormous progress in the development of novel technologies for DNA sequencing. The advent of next-generation sequencing technologies now makes it possible to sequence an entire human genome in one or a few experiments. As a consequence, several individual human genomes have now been fully sequenced, using different experimental strategies. Although the protocols differ between the various sequencing technologies, the challenges of analyzing the data, calling variation, and interpreting the results are similar for all platforms. Here, we give an overview of the human genome sequencing projects completed to date. The strategies for aligning sequence reads and extracting information about different types of genetic variation from the sequence data are discussed. Identification of structural variation, such as copy number variation and insertion-deletion variants, can be complex, and there are a plethora of algorithms and analysis tools available. We also give an overview of the challenge of interpreting the whole-genome sequence data both from a technical and clinical perspective.

Teamwork and leadership in cardiopulmonary resuscitation.

Despite substantial efforts to make cardiopulmonary resuscitation (CPR) algorithms known to healthcare workers, the outcome of CPR has remained poor during the past decades. Resuscitation teams often deviate from algorithms of CPR. Emerging evidence suggests that in addition to technical skills of individual rescuers, human factors such as teamwork and leadership affect adherence to algorithms and hence the outcome of CPR. This review describes the state of the science linking team interactions to the performance of CPR. Because logistical barriers make controlled measurement of team interaction in the earliest moments of real-life resuscitations challenging, our review focuses mainly on high-fidelity human simulator studies. This technique allows in-depth investigation of complex human interactions using precise and reproducible methods. It also removes variability in the clinical parameters of resuscitation, thus letting researchers study human factors and team interactions without confounding by clinical variability from resuscitation to resuscitation. Research has shown that a prolonged process of team building and poor leadership behavior are associated with significant shortcomings in CPR. Teamwork and leadership training have been shown to improve subsequent team performance during resuscitation and have recently been included in guidelines for advanced life support courses. We propose that further studies on the effects of team interactions on performance of complex medical emergency interventions such as resuscitation are needed. Future efforts to better understand the influence of team factors (e.g., team member status, team hierarchy, handling of human errors), individual factors (e.g., sex differences, perceived stress), and external factors (e.g., equipment, algorithms, institutional characteristics) on team performance in resuscitation situations are critical to improve CPR performance and medical outcomes of patients.

Perspective: medical education research and the institutional review board: reexamining the process.

Medical school and residency training curricula across the country have undergone extensive revisions and, much like clinical quality improvement (QI) initiatives, require assessments of new programs. Because sharing knowledge is a hallmark of academic medicine, program evaluation may come under the purview of the institutional review board (IRB); however, the distinction between QI and research is often unclear. And yet a medical education (ME) inquiry can be designed according to either paradigm. The purpose of this article is to bring IRBs and ME researchers closer to a shared understanding of key concepts underlying human participation in research and QI activities, and to consensus on the application of these concepts. The current QI discourse provides a useful framework for making this distinction; the authors identify key theoretical principles and practical considerations derived from this work that are relevant to ME and training, such as the application of the regulatory definition of human subject research to ME inquiries. For ME inquiries defined as human subject research, and therefore subject to IRB review, this article explores the application of the human research regulations to ME research. It concludes with practical suggestions for institutions, IRBs, and ME researchers, which range from formal procedures for making the QI versus research distinction, to instruction in study design and development and the human subject regulatory implications. The intent is to promote a discussion that will result in greater consensus and a more consistent application of the regulatory framework.

Characterization of copy number-stable regions in the human genome.

In the past few years the number of copy number variants (CNVs) identified in the human genome has increased significantly, but our understanding of the functional impact of CNVs is still limited. Clinically significant variations cannot easily be distinguished from benign, complicating interpretation of patient data. Multiple studies have focused on analysis of regions that vary in copy number in specific disorders. Here we use the opposite strategy and focus our analysis on regions that never seem to vary in the general population, hypothesizing that these are copy number stable because variations within them are deleterious. Our results show that copy number stable regions are characterized by correlation with a number of genomic features, allowing us to define a list of genomic regions that are dosage sensitive in humans. We find that these dosage-sensitive regions show significant overlap with de novo CNVs identified in patients with intellectual disability or autism. There is also a significant association between copy number stable regions and rare inherited variants in autism patients, but not in controls. Based on this predictive power, we propose that copy number stable regions can be used to complement maps of known CNVs to facilitate interpretation of patient data.

Protein contents in biological membranes can explain abnormal solvation of charged and polar residues.

Transmembrane helices are generally believed to insert into membranes based on their hydrophobicity. Nevertheless, there are important exceptions where polar residues have great functional importance, for instance the S4 helix of voltage-gated ion channels. It has been shown experimentally that insertion can be accomplished by hydrophobic counterbalance, predicting an arginine insertion cost of only 2.5 kcal/mol, compared with 14.9 kcal/mol in cyclohexane. Previous simulations of pure bilayers have produced values close to the pure hydrocarbon, which has lead to spirited discussion about the experimental conditions. Here, we have performed computer simulations of models better mimicking biological membranes by explicitly including protein helices at mass fractions from 15% to 55%, as well as an actual translocon. This has a striking effect on the solvation free energy of arginine. With some polar residues present, the solvation cost comes close to experimental observation at approximately 30% mass fraction, and negligible at 40%. In the presence of a translocon in the membrane, the cost of inserting arginine next to the lateral gate can be as low as 3-5 kcal/mol. The effect is mainly due to the extra helices making it easier to retain hydration water. These results offer a possible explanation for the discrepancy between the in vivo hydrophobicity scale and computer simulations and highlight the importance of the high protein contents in membranes. Although many membrane proteins are stable in pure bilayers, such simplified models might not be sufficiently accurate for insertion of polar or charged residues in biological membranes.

The role of lipid composition for insertion and stabilization of amino acids in membranes.

While most membrane protein helices are clearly hydrophobic, recent experiments have indicated that it is possible to insert marginally hydrophobic helices into bilayers and have suggested apparent in vivo free energies of insertion for charged residues that are low, e.g., a few kcals for arginine. In contrast, a number of biophysical simulation studies have predicted that the bilayer interior is close to a pure hydrophobic environment with large penalties for hydrophilic amino acids--and yet the experimental scales do significantly better at predicting actual membrane proteins from sequence. Here, we have systematically studied the dependence of the free energy profiles on lipid properties, including tail length, saturation, headgroup hydrogen bond strength, and charge, both to see to whether the in vivo insertion can be explained in whole or part from lipid composition of the endoplasmic reticulum (ER) membranes, and if the solvation properties can help interpret how protein function depends on the lipids. We find that lipid charge is important to stabilize charged amino acids inside the bilayer (with implications, e.g., for ion channels), that thicker bilayers have higher solvation costs for hydrophilic side chains, and that headgroup hydrogen bond strength determines how adaptive the lipids are as a hydrophobic/hydrophilic solvent. None of the different free energy profiles are even close to the low apparent in vivo insertion cost, which suggests that regardless of the specific ER membrane composition the current experimental results cannot be explained by normal lipid-type variation.

Titratable amino acid solvation in lipid membranes as a function of protonation state.

Knowledge about the insertion and stabilization of membrane proteins is a key step toward understanding their function and enabling membrane protein design. Transmembrane helices are normally quite hydrophobic so as to efficiently insert into membranes, but there are many exceptions with polar or titratable residues. An obvious example is the S4 helices of voltage-gated ion channels with up to 4 arginines, leading to vivid discussion about whether such helices can insert spontaneously, and if so, what their conformation, protonation state, and cost of insertion really are. To address this question, we have determined geometric and energetic solvation properties for different protonation states of the titrateable amino acids, including hydration, side chain orientation, free energy profiles, and effects on the membrane thickness. As expected, charged states are significantly more expensive to insert (8-16 kcal/mol) than neutral variants (1-3 kcal/mol). Although both sets of values exhibit quite high relative correlation with experimental in vivo hydrophobicity scales, the magnitudes of the in vivo hydrophobicity scales are much lower and strikingly appears as a compressed version of the calculated values. This agrees well with computational studies on longer lipids but results in an obvious paradox: the differences between in vivo insertion and simulations cannot be explained by methodological differences in force fields, possible limited hydrophobic thickness of the endoplasmic reticulum (ER) membrane, or parameters; even anionic lipid head groups (PG) only have limited effect on charged side chains, and virtually none for hydrophobic ones. This leads us to propose a model for in vivo insertion that could reconcile these differences and explain the correlation: if there are considerable hydrophobic barriers inside the translocon, the experimental reference state for the solvation free energy when comparing insertion/translocation in vivo would be quite close to the bilayer environment rather than water.

Position-resolved free energy of solvation for amino acids in lipid membranes from molecular dynamics simulations.

Studies of insertion and interactions of amino acids in lipid membranes are pivotal to our understanding of membrane protein structure and function. Calculating the insertion cost as a function of transmembrane helix sequence is thus an important step towards improved membrane protein prediction and eventually drug design. Here, we present position-dependent free energies of solvation for all amino acid analogs along the membrane normal. The profiles cover the entire region from bulk water to hydrophobic core, and were produced from all-atom molecular dynamics simulations. Experimental differences corresponding to mutations and costs for entire segments match experimental data well, and in addition the profiles provide the spatial resolution currently not available from experiments. Polar side-chains largely maintain their hydration and assume quite ordered conformations, which indicates the solvation cost is mainly entropic. The cost of solvating charged side-chains is not only significantly lower than for implicit solvation models, but also close to experiments, meaning these could well maintain their protonation states inside the membrane. The single notable exception to the experimental agreement is proline, which is quite expensive to introduce in vivo despite its hydrophobicity--a difference possibly explained by kinks making it harder to insert helices in the translocon.